Mediating ERK1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome (original) (raw)
Expression of Q79R in the cardiomyocytes leads to different outcomes depending upon the developmental time. To investigate the gain-of-function effect of the NS SHP2 mutation on the heart, we generated Tg mice in which normal, WT and mutated SHP2 was expressed specifically in cardiomyocytes, either during gestation or after birth. The most frequently found mutation in the N-SH2 domain of SHP2, Q79R (17), which has a 4.5-fold higher phosphatase activity than that of WT SHP2 (19), was used for this study. The β– and α–myosin heavy chain (β-MHC and α-MHC) promoters were used to express WT and Q79R SHP2 in the fetal and postnatal ventricles, respectively (Figure 1, A and B). Multiple lines were established (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI30756DS1), and, after expression levels were determined for each Tg construct, we selected single lines of mice showing roughly equivalent increases in cardiac SHP2 protein content for subsequent analyses (Figure 1C). To confirm the developmental segregation of the promoters — that is, inactivity of the α-MHC promoter during ventricular development — total SHP2 protein was measured from E9.5 through E16.5 in the α-MHC Q79R line shown in Figure 1C. As expected, SHP2 protein levels did not differ between the nontransgenic (Ntg) and Tg animals (Figure 1D).
Developmental stage–specific transgene expression of normal or mutant SHP2 in the ventricles. (A) Schematic diagram of Ptpn11 showing the approximate exon boundaries for the functional domains and location of the Q79R mutation. (B) Constructs used to generate cardiomyocyte-specific expression of normal (WT) or NS mutant Q79R protein. Mouse cDNA containing either normal (WT) or mutant (Q79R) SHP2 was linked to either the β- or α-MHC promoter to express the transgene. The β-MHC promoter expresses the transgene in the ventricular cardiomyocytes between E9.25 and E19, while the α-MHC promoter expresses the transgene in the postnatal ventricle. Vertical boxes in β-MHC and α-MHC promoters represent noncoding exons in 5′-untranslated region. A poly adenylation signal sequence derived from the human growth hormone gene (hGH) was inserted downstream of the cDNA. (C) Western blot analyses of Tg cardiac tissue. In β-MHC hearts, SHP2 protein expression was increased 5.3-fold in both the WT and Q79R Tg hearts at E16.5. SHP2 protein expression was increased 11.4- and 13.6-fold in the α-MHC WT and Q79R hearts, respectively, at 3 months after birth. (D) To confirm that the α-MHC–driven SHP2 construct was transcriptionally inactive during gestation, total SHP2 protein in the Q79R line (13.6-fold overexpression after birth) was measured at E9.5–E16.5, the period during which compaction occurs. When values were normalized to a GAPDH loading control, no statistically significant increases in protein levels were observed.
At E16.5, ventricular noncompaction, ventricular septal defects (VSDs), and abnormal anatomy of the interventricular groove were prominent in the β-MHC Q79R hearts, a phenotype similar to that reported in another mouse model of NS, in which the D61G SHP2 mutation was expressed (18). When multiple lines showing varying degrees of overexpression were analyzed, the severity and frequency of the defects were both dose dependent (Supplemental Figure 1). In contrast, α-MHC WT, α-MHC Q79R, and β-MHC WT hearts showed no aberrant ventricular architecture (Figure 2A). Cardiac histology at 3 months showed that significant SHP2 overexpression in the postnatal cardiomyocytes had no effects in either the α-MHC WT or Q79R hearts. In the β-MHC Q79R hearts, embryonic ventricular noncompaction persisted postnatally (Figure 2B), with no evidence of fibrosis (Figure 2C).
Histological findings. (A) Four-chamber view of the hearts at E16.5 (original magnification, ×4). Ventricular noncompaction, VSDs (arrowheads), and abnormal anatomy of the interventricular groove (*) are prominent in the β-MHC Q79R hearts, while the α-MHC WT and Q79R and β-MHC WT hearts appear to be normal. (B) Longitudinal ventricular sections at 3 months after birth, stained with H&E (original magnification, ×4). Each image shows the right-ventricular free wall on the left and the ventricular septum on the right. Despite SHP2 overexpression in the postnatal cardiomyocytes, no abnormalities were seen in the α-MHC WT and Q79R hearts. In the β-MHC Q79R hearts, the ventricular noncompaction observed in the embryonic stage continued to present postnatally. (C) Masson trichrome staining of the right-ventricular free walls from the rectangular area indicated in B (original magnification, ×10). For each panel at least 5 hearts were analyzed. For all panels, orientation is apical on the right and basal on the left. (D) Representative photograph of an end-stage failing heart in the β-MHC Q79R mouse (6 weeks old; original magnification, ×2.8). Compared with those of the Ntg littermates, β-MHC Q79R hearts showed both atrial and ventricular dilatation and abnormal interventricular groove anatomy (*). Left-ventricular hypofunction caused regurgitation toward the left atrium, resulting in massive thrombi. (E and F) Analyses of heart weight to body weight (HW/BW) ratio and lung weight to body weight (LW/BW) ratio in age- and sex-matched mice (3 months after birth, n = 8 [4 male, 4 female]). HW/BW and LW/BW ratios were increased in β-MHC Q79R mice. *P < 0.005.
We further analyzed the effect of SHP2 expression on cardiac function and fate in these mice. Echocardiography revealed that β-MHC Q79R hearts showed markedly decreased contractility and ventricular wall thickness, while cardiac function was normal in α-MHC WT, α-MHC Q79R, and β-MHC WT mice (Table 1). Cardiac hypofunction in β-MHC Q79R mice resulted in congestive heart failure (Figure 2D). Animals typically presented postmortem with atrial and ventricular dilatation, chronic left atrial thrombi, and abnormal anatomy of the intraventricular groove. Heart weight to body weight (HW/BW) ratios and lung weight to body weight (LW/BW) ratios were each increased (Figure 2, E and F). In β-MHC Q79R mice, 38.8% (19 of 49) died by 8 months, while all Ntg (n = 32), α-MHC WT (n = 37), α-MHC Q79R (n = 50), and β-MHC WT (n = 32) mice survived.
Cardiac function in α- and β-MHC SHP2 mice
Q79R SHP2 expression selectively activates ERK1/2. SHP2 regulates intracellular signaling pathways that help control cell proliferation, differentiation, migration, adhesion, and survival (15) by modulating MAPK cascades (21–24). We thus examined several candidate downstream signaling pathways for activation by the Q79R SHP2 mutation during gestation. Consistent with previous in vitro data (19), the ERK1/2 branch was hyperphosphorylated in β-MHC Q79R hearts (Figure 3A and Supplemental Table 1) but not in α-MHC Q79R hearts (data not shown) during gestation, consistent with the activities of the 2 promoters during cardiac development (25). ERK1/2 hyperactivation was sustained throughout the latter half of gestation in the β-MHC Q79R hearts (Figure 3, B–D). We further examined ERK1/2 in the adult β-MHC Q79R hearts and asked whether expression varied in accordance with cardiac dysfunction. Interestingly, ERK1/2 activation was downregulated postnatally even in the failing hearts (Figure 3B, right). ERK1/2 was hyperphosphorylated in the α-MHC Q79R hearts at 3 months (Figure 3A), suggesting that the signaling activity parallels SHP2 expression, but it appears that chronic activation of this pathway in cardiomyocytes after cardiac organogenesis is largely benign, as these mice had normal lifespans, conserved cardiac function (Table 1), and no pathology (Figure 2). The other MAPK cascades, including p38, JNK1/2, and ERK5, were not activated (Figure 3A and Supplemental Table 1). It has been reported that SHP2 can also participate in cell survival, migration, adhesion, and myogenesis by signaling through the Akt, JAK/STAT, or RhoA pathways (26–29). However, the expression of these proteins or the respective effectors (Akt, STAT1, -3, -5, and MYPT) were all unchanged in the cardiomyocyte population (Figure 3A and Supplemental Table 1).
SHP2 Q79R expression results in ERK1/2 hyperphosphorylation. (A) Western blots of the signaling pathways potentially downstream of SHP2. ERK1/2 is hyperphosphorylated in β-MHC Q79R (E16.5) and α-MHC Q79R (3-month) hearts. The phosphorylated species were quantitated and the values given in Supplemental Table 1. (B) ERK1/2 is hyperphosphorylated throughout the latter half of gestation in the β-MHC Q79R hearts but this does not occur postnatally, when the β-MHC promoter is inactive. All lanes contained pooled protein from at least 5 hearts. Quantitation of the blots is shown in the Supplemental Table 1. (C and D) Quantitation of p-ERK1 and p-ERK2, respectively. The blots shown in B were quantitated using a Storm PhosphorImager, as described in Methods. CHF, congestive heart failure; NF, nonfailing.
Reduction of ERK1/2 hyperphosphorylation in β-MHC Q79R hearts. Both ERK1 and ERK2 are widely expressed throughout development (30). ERK1/2 activation is required for embryogenesis (31), with the duration and magnitude of ERK activity differing among cell types (32). In the mouse, brief pulses of ERK1/2 activation occur in the heart primordia, suggesting that ERK1/2 signaling plays a transient and dynamic role in cardiac development, although the specific role of each ERK isoform remains unclear (31). Studies with ERK1- and ERK2-knockout mice showed that ablation of one ERK did not influence the expression of the other ERK isoform, suggesting at least some degree of autonomy (30, 33). We hypothesized that sustained cardiomyocyte ERK1/2 hyperphosphorylation during embryogenesis had specific adverse effects on cardiac maturation, leading to ventricular noncompaction and VSDs. To establish the sufficiency of ERK1/2 hyperphosphorylation’s effects on cardiac development, we crossed the β-MHC Q79R mice to _Erk1_- and _Erk2_-knockout mice, respectively. Homozygous _Erk1_-null (Erk1–/–) mice are viable and fertile, although thymocyte proliferation and maturation are affected (33). Homozygous loss of Erk2 (Erk2–/–) results in embryonic lethality, but the heterozygotes survive into adulthood (30). Thus, we generated β-MHC Q79R × Erk1–/– and β-MHC Q79R × Erk2+/– mice. At E16.5, ERK1 hyperactivation was completely ablated in the β-MHC Q79R × Erk1–/– hearts, and ERK2 hyperactivation was substantially reduced in the MHC Q79R × Erk2+/– hearts (Figure 4, A and B). Similar results were observed at E11.5 and E13.5 as well (data not shown). In both β-MHC Q79R × Erk1–/– and β-MHC Q79R × Erk2+/– fetal hearts (E16.5), the ventricular noncompaction and concomitant thinning of the compact myocardium found in the β-MHC Q79R hearts were significantly decreased (Figure 4C). These changes are more readily apparent at higher magnification (Figure 4D). Qualitative observations were confirmed by quantitation (Figure 4E), and the frequency of VSDs at E16.5 was also decreased: Ntg (n = 10), 0 VSDs; Erk1–/– (n = 9), 1 VSD; Erk2+/– (n = 10), 0 VSDs; β-MHC Q79R (n = 10), 7 VSDs; β-MHC Q79R × Erk1–/– (n = 11), 2 VSDs; β-MHC Q79R × Erk2+/– (n = 10), 1 VSD). The data indicate that both ERK1 and ERK2 activation are required for the full phenotype to present.
Reduction of ERK1/2 hyperphosphorylation rescues Q79R-induced heart disease. Western blot analysis (A) of E16.5 hearts and quantitation (B). ERK1 and ERK2 hyperphosphorylation in the β-MHC Q79R heart is reduced after crossing to the Erk1–/– and Erk2+/– mice. (C) Histology of the E16.5 hearts shows that the ventricular noncompaction and concomitant wall thinning seen in β-MHC Q79R hearts are ameliorated when the mice are crossed to the Erk1–/– and Erk2+/– mice. (D) Higher magnifications of a subsection of the hearts shown in C. Identically placed, small areas derived from the apical side of the right-ventricular free wall were selected and clearly show the thinning present in the Q79R mutants, compared with either the WT hearts or the hearts derived from the Erk1–/– and Erk2+/– crosses. (E) Quantitation of ventricular compaction at E16.5. The compaction index was determined using Metamorph software (version 6.3) as described in Methods. RV, right ventricle; LV, left ventricle. *P < 0.0001 in RV; **P < 0.0001 in LV (n = 7). Vent., ventricular.
We then analyzed the histology and cardiac function of postnatal hearts derived from these crosses to see whether the rescue persisted. In β-MHC Q79R × Erk1–/– and β-MHC Q79R × Erk2+/– mice, ventricular noncompaction was rescued in both the fetal and adult hearts (Figure 5A). Echocardiography at 15 weeks demonstrated significant improvements in both ventricular wall thickness and contractility (Table 2), with HW/BW and LW/BW ratios indistinguishable from those of Ntg hearts (Figure 5B). The data show that reduction of inappropriate ERK1 or ERK2 hyperphosphorylation during embryogenesis restores normal cardiac anatomy and function.
Analyses of adult hearts. (A) Histology of 3-month-old hearts stained with Masson trichrome (right-ventricular free wall; original magnification, ×10). Rescued ventricular noncompaction observed in the embryonic stage persists postnatally in the β-MHC Q79R × Erk1–/– and β-MHC Q79R × Erk2+/– hearts. All samples were taken from the identical location in the right ventricle, as denoted by the rectangle in Figure 2B. (B) HW/BW and LW/BW ratios in age- and sex-matched mice (3 months after birth; n = 8 [4 male, 4 female]). *P < 0.05.
Reduction of ERK1/2 hyperphosphorylation restores cardiac function
ERK not only mediates cell survival but also regulates cell differentiation and proliferation (32). We observed no changes in caspase-3 activity or in the levels of the terminal differentiation markers α- and β-MHC (Supplemental Figure 2). Therefore, we hypothesized that ERK1/2 hyperactivation affected cardiomyocyte cell cycling or proliferation. In β-MHC Q79R hearts, enhanced staining with the cell proliferation marker Ki-67 was apparent, particularly in the noncompacted ventricle (Figure 6A) and in the cells located at the boundaries of the muscular ventricular septum and atrioventricular cushion tissue (Figure 6B). However, normal levels of Ki-67 were restored in the β-MHC Q79R × Erk1–/– and β-MHC Q79R × Erk2+/– hearts (Figure 6, A–C). These data suggest that enhanced cardiomyocyte proliferation or alterations in cell-cycling kinetics through ERK1/2 hyperactivation unfavorably impact normal ventricular maturation and that ablation of abnormal ERK1/2 activation can prevent the occurrence of Q79R-mediated cardiac malformations.
Reduction of ERK1/2 hyperphosphorylation inhibits enhanced cardiomyocyte proliferation. (A and B) Ki-67 immunostaining (E16.5; original magnification, ×20). Ki-67–positive cardiomyocytes are abundant in the β-MHC Q79R hearts, particularly in the trabecular myocardium (A) and at the tip of the muscular ventricular septum (B, arrowheads). Normal cardiomyocyte proliferation is restored in the Erk1–/– and Erk2+/– backgrounds. (C) Quantitation of Ki-67–positive cells (n = 5). *P < 0.0001. A total of 4,500 cells were counted in left, right, and septal areas of 5 hearts from each group.